Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.
OBJECTIVE
To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts.


METHODS
Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release.


RESULTS
Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment.


CONCLUSIONS
Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.
Crowston
J G
JG
Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom.
Akbar
A N
AN
Constable
P H
PH
Occleston
N L
NL
Daniels
J T
JT
Khaw
P T
PT
D000963:Antimetabolites / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002452:Cell Count; D002478:Cells, Cultured; D003238:Connective Tissue / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology*
D000963:Antimetabolites; D016685:Mitomycin; D007770:L-Lactate Dehydrogenase; D005472:Fluorouracil